This disclosure pertains to oligonucleotide-quencher conjugates with improved fluorescence characteristics, and to reagents suitable for incorporating novel quencher moieties into oligonucleotides. The disclosure also pertains to the use of oligonucleotide-quencher conjugates in detection methods for nucleic acid targets.
Minor groove binding (MGB) and fluorescence quenching are two key features of fluorescence-based DNA probe technologies. The first feature provides improved duplex stabilization and hybridization specificity, whereas the second one is required for reduction of background fluorescence of unhybridized probes. Historically, two independent functional groups have been used to fulfill these functions. The MGB and quenchers have been introduced into DNA probes using two reagents such as MGB-modified DNA synthesis solid supports and Quencher phosphoramidites. In another approach a MGB-Quencher solid support is utilized wherein MGB and quencher are linked together via a flexible spacer with each group serving its unique function. The existing MGB-Quencher structures and respective DNA synthesis reagents as disclosed in U.S. Pat. No. 6,492,346 are complicated and costly to manufacture.